A. Affinity chromatography (AC) followed by size exclusion chromatography (SEC)

B. AC only

C. AC followed by ion-exchange (IEX) followed by SEC

D. AC followed by IEX, followed by hydrophobic interaction (HIC) and then SEC

Protein Purification Strategy: Affinity Chromatography Followed by Size Exclusion Chromatography

For high-purity target protein from E. coli overexpressing an affinity-tagged version, the optimal strategy balances efficiency and final polishing. The correct answer is A. Affinity chromatography (AC) followed by size exclusion chromatography (SEC).

Why This Question Matters

This multiple-choice question tests core biochemistry knowledge for recombinant protein purification, common in GATE Life Sciences and molecular biology research. Starting from E. coli lysate with an affinity tag (like His-tag or GST), the goal is high purity for applications like crystallography or assays. Standard protocols use orthogonal steps: capture via specific binding, then polishing.

Option Breakdown

Each option builds on AC as the capture step, exploiting the affinity tag for quick, high-yield isolation from crude lysate.

• A. AC followed by SEC: Best choice. AC captures >90% pure target via tag-specific binding (e.g., Ni-NTA for His-tag). SEC polishes by size, removing aggregates, fragments, and minor contaminants while exchanging buffer. SEC acts as a “polishing” step, ideal for final high purity without overcomplicating.

• B. AC only: Often 80-95% pure but insufficient for “high purity” needs. Residual E. coli proteins, tag-cleaving protease byproducts, or multimers remain. Fine for quick preps, but not for demanding apps.

• C. AC followed by IEX followed by SEC: Overkill for most cases. IEX (charge-based) adds an intermediate step, good if post-AC impurities share size but differ in pI. Increases time, dilution, and risk of low yield; unnecessary when SEC alone suffices.

• D. AC followed by IEX, HIC, and SEC: Excessive multi-step process. HIC (hydrophobicity-based) suits high-salt conditions or surface hydrophobics, but four steps dilute protein, raise costs, and risk instability. Used only for ultra-tricky proteins.

Standard Workflow

1. Lyse E. coli, clarify supernatant.

2. AC: Bind/elute via tag (e.g., imidazole gradient).

3. SEC: Run on Superdex column; target elutes by native size, separating monomers from dimers/aggregates.

This AC-SEC combo achieves >95% purity efficiently, matching industrial and academic standards for tagged proteins.